HAC Vector
Conventional vectors, such as plasmid, cosmid, bacteria artificial chromosome (BAC), yeast artificial chromosome (YAC), or virus vectors, which are commonly used when introducing genes into mammalian cells, suffer from the problem of transient expression of the introduced genes in the case of episomes. When genes are integrated into chromosomes of host cells, however, such conventional vectors suffer from a number of problems, such as the host chromosome genes being disrupted, the copy number of genes introduced not being regulated, the introduced genes being inactivated, or gene expression being affected by a control sequence of the host chromosome into which the genes have been integrated.
As a means for resolving such problems, the research group consisting of the present inventors constructed a novel HAC (21HAC) vector system with the use of human chromosome 21 as a starting material, from which unnecessary genes with apparent structures have been removed, and which is capable of easy introduction of foreign DNA (WO 2004/031385; Katoh et al., Biochem Biophys Res Commun; 321: 280-290, 2004). In these documents, the present inventors demonstrated that the 21HAC vector is capable of autonomous replication and distribution in mammalian host cells, including humans, the 21HAC vector is retained stably, and a given copy number of genes could be introduced, without disrupting host chromosomes. They also demonstrated that the 21HAC vector into which a therapeutic gene, human erythropoietin (hEPO), had been introduced as a model intended for ex vivo hormone-replacement gene therapy could be introduced into cultured normal primary human fibroblasts, EPO expression could be maintained for a long period of time, the expression level thereof would be suppressed to a level approximately 1/1,000 of that in CHO cells, cultured normal primary human fibroblasts into which HAC had been introduced could be amplified from a single colony to a maximum of 3.8×107 cells, and 65.5% to 90.9% of the genes could be retained after 6- or 9-passage culture under non-selective conditions (WO 2004/031385; Kakeda et al., Gene Therapy; 12: 852-856, 2005).
In the case of a baboon model of ex vivo EPO-replacement gene therapy, it is reported that hypodermic transplantation of capsules of cultured primary baboon MSCs (0.81 to 6.6×106 cells/kg) into which hEPO genes had been introduced with the use of a retrovirus vector resulted in enhanced blood EPO level and recovery from anemia (Bartholomew, A. et al., Hum Gene Ther; 12: 1527-1541, 2001). For the purpose of gene therapy, however, further improvement is desired in expression levels per cell. From the viewpoint of the limit of division of cultured normal primary human fibroblasts, the number of instances of cell division is preferably small. In order to effectively amplify cells into which therapeutic HACs have been introduced that can exhibit effects in vivo, stability of vectors should be improved and enhanced.
An example of a means for resolving the aforementioned problem is to construct an HAC vector having a functional telomere, with the use of a highly stable human chromosome as a human chromosome that acts as a basic backbone of the vector.
It is known that expression of foreign genes is influenced by the chromatin structure or the presence or absence of the control sequence in the vicinity of the insertion site. It is also known that a functional telomere of a sufficient length is necessary in order to stably retain the chromosome (Smogorzewska, A. et al., Anuu. Rev. Biochem., vol. 73, pp. 177-208, 2004). While there is an example in which an insulator had been used in order to eliminate the influence of the chromatin structure or the control sequence on HAC (Otsuki et al., Biochem Biophys Res Commun; 329: 1018-1025, 2005), elimination of the influence of the chromatin structure or the control sequence has not yet been examined, and such insulator would not regulate the telomere sequence. Also, improvement of stability of HAC vectors by telomere sequence regulation, such as addition of a telomere sequence or restoration of telomere length with the aid of telomerase on HAC, has not yet been examined.
Improvement of Efficiency of Homologous Recombination
When introducing foreign genes, drug-resistant genes are generally introduced in order to select cells into which the genes of interest have been introduced. In many cases, drug-resistant genes are positioned in parallel with the target foreign genes in the same vector. When a plurality of gene expression units are arrayed in parallel, it is known that they interfere with each other and the expression levels of the foreign genes are lowered. Up to the present, a drug-resistant gene is positioned as an exon, spliced, and expressed to lower drug resistance, and a cell in which the introduced gene is expressed at a high level, is selected in order to elevate the expression levels, when constructing high-level expression/production cells (Barnett, R. S. et al., Antibody expression and engineering, Chapter 3, p. 27-40, American Chemical Society, 1995). However, there are no reports regarding significantly improved expression of foreign genes resulted from elimination of drug-resistant genes.
Removal of drug-resistant genes located downstream is known to improve the efficiency of homologous recombination in mouse ES cells, when knocking in a foreign gene in the antibody light-chain κ locus (JP Patent Publication (kokai) No. 2006-94849 (A)), although this is not intended to improve foreign gene expression.
Stability of Human Chromosome and Gene Expression
As a result of experiments involving mouse ES cells, it is known that a larger chromosome fragment introduced into a cell results in a lower contribution ratio of such cell in an individual chimera. The research group consisting of the present inventors demonstrated that an individual chimera could be prepared by introducing human chromosome 14, 2, and 22 fragments, including antibody genes, into mouse embryonic stem cells, the introduced antibody genes are functionally expressed in the individuals, human chromosome fragments are retained stably in an individual chimera, and such genes can be inherited by the following generation through a germ line (Tomizuka et al., Nature Genet., U.S.A., vol. 16, pp. 133-143, 1997; and Tomizuka et al., P.N.A.S., U.S.A., vol. 97, pp. 722-727, 2000). The naturally-occurring fragment, SC20, derived from human chromosome 14, which had been isolated so as to prepare a mouse carrying a human antibody heavy chain gene, and HAC prepared by cloning a human chromosome 22 region comprising an antibody light chain gene into SC20 were examined in terms of stability and germ-line transmission in a mouse (Kuroiwa et al., Nature Biotech., U.S.A., vol. 18, pp. 1086-1090, 2000). Further, the present inventors have demonstrated that the above SC20-derived HAC would be retained stably in a cloned bovine individual prepared by somatic nuclear transplantation and that the introduced antibody genes would be functionally expressed in individuals (Kuroiwa et al., Nature Biotech., U.S.A., vol. 20, pp. 889-894, 2002). The above human chromosome-14-derived naturally-occurring fragment and HAC have not yet been examined in terms of stability and expression in cultured normal primary human cells or in human cell lines. Further, other human chromosome-derived naturally-occurring fragments and HACs, except for human chromosome 21, have not yet been fully examined in terms of stability and expression in cultured normal primary human cells or in human cell lines. While there is an example of a report in which the human cell line retains minichromosomes derived from human chromosome X (Mills et al., Hum. Mol. Genet., U.K., vol. 8, pp. 751-761, 1999), such minichromosomes are found to result in a significant number of multicopies after subculture, and maintenance of a given number of copies still remains difficult.
Regulation of Expression and Stability by Telomere
A telomere is a repeat sequence located at the end of a chromosome, and it is conserved in a wide range of organism species from yeast cells to mammalian cells, including humans. In a normal human cell, repeats of TTAGGG reach a size of 15 to 0.4 kb. A telomere becomes shorter as cell division proceeds, and heterochromosomes (i.e., involving fusion, drop out, split, alteration of chromosome number, or the like) appear or increase as the telomere length becomes shorter. Thus, a telomere is considered to be necessary for protection and maintenance of stability of chromosomes (Smogorzewska, A. et al., Anuu. Rev. Biochem., vol. 73, pp. 177-208, 2004; and Ning, Y et al., Hum Mol Genet., vol. 12, pp. 1329-1336, 2003). Thus, a functional telomere structure of the sufficient length is considered to result in improved stability of chromosomes.
A telomere length can be artificially extended by forced expression of telomerase or deletion of a telomere-binding protein (POT•TRF2) (Crisrofari, C. et al., EMBO J, vol. 25, pp. 565-574, 2006; Smogorzewska, A. et al., Anuu. Rev. Biochem., vol. 73, pp. 177-208, 2004). These techniques verified the increased number of instances of cell division and prolonged life in cultured normal primary human cells and in human cell lines; however, whether or not chromosome stability can be restored and improved has not yet been elucidated. When endogenous telomerase activity was elevated after crisis and the shortened telomerase was extended and maintained at a given length, the frequency at which heterochromosomes appearing in immortalized cells was constant without increase or decrease (Counter, C. M. et al., EMBO J, vol. 11, pp. 1921-1929, 1992). In this case, however, restoration and improvement of chromosome stability were not achieved.
A subtelomere is a chromosome region of approximately 300 to 500 kb, which is located adjacent to the centromere side of the telomere (TTAGGG)n repeat sequence at the chromosome terminus, and which has a segmented/overlapped domain or a (TTAGGG)n-like repeat sequence. Because of the high degree of homology of the segmented/overlapped domain structure, it is known that displacement or recombination is highly likely to occur within the subtelomere of the same chromosome or between subtelomeres of different chromosomes (Mefford, H. C. et al., Nat Rev Genet., vol. 3, pp. 91-102, 2002; Riethman, H et al., Chromosome Res., vol. 13, pp. 505-515, 2005; Linardopoulou, E. V. et al., Nature, vol. 437, pp. 94-100, 2005). The subtelomere length of the long arm of human chromosome 14 is identified as a 499-bp segmented/overlapped domain, and the presence of an unidentified sequence of 20 kb or shorter between the telomere sequence (TTAGGG)n and the subtelomeric region is suggested (Riethman H et al., Genome Res., vol. 14, pp. 18-28, 2004). As subtelomere functions, for example, restoration and maintenance of the telomere via recombination acceleration utilizing high homology in the absence of telomerase, adaptation to a new environment due to subtelomere plasticity, and induction of displacement and deletion that cause diseases are suggested, although details thereof have not yet been elucidated.
Regarding the correlation between the telomere and gene expression or the correlation between the telomere and the subtelomere and gene expression, it is observed that expression of the introduced foreign gene in a proximal site of the telomere is affected by silencing by heterochromatinization (i.e., telomere position effect (TPE)) in cells having a chromosome in which the terminus has been substituted with the telomere sequence upon random insertion of the telomere sequence (Tham W H et al., Oncogene, vol. 21, pp. 512-521, 2002). Regarding TPE, it is reported that telomere extension is correlated with silencing (Baur J A et al., Science, vol. 292, pp. 2075-2077, 2001). Regarding expression of genes of endogenous telomeric regions, however, there was no correlation between a gene expression pattern and decrease in the telomere length or the distance from the telomere end in neonatal human foreskin fibroblasts before and after subculture (Ning Y et al., Hum Mol Genet., vol. 12, pp. 1329-1336, 2003). Thus, discussion regarding the correlation between the telomere and gene expression has not yet been concluded.